The objective of the proposed research is the characterization of the primary incision step in excision repair of human cell DNA. An endonuclease that is specific for UV irradiated DNA, but which does not apparently recognise UV induced pyrimidine dimers will initially be studied in crude cell extracts. The spectrum of lesions recognized by the endonuclease will be determined with DNA which has been treated with various oncolytic and carcinogenic alkylating agents, or with ionising radiation. Endonuclease activity will be assayed by following the conversion of superhelical PM2 viral DNA to the relaxed circular form. Precursor and product DNA will be separated by velocity gradient centrifugation. Conclusive characterization of the enzyme's substrate specificity cannot be achieved with a crude extract. A more rapid assay, will be adopted to facilitate enzyme purification. Characterization for substrate specificity will continue throughout the purification to detect the possible separation of endonucleases with different specificities. With purified endonuclease of defined substrate specificity a method will become available for determination of lesions in cellular DNA. The in vivo kinetics of repair in human cells can then be studied by measuring the disappearance of endonuclease recognition sites from intracellularly damaged DNA.